With the exception the binding site of the C-terminal "headpiece" domain of villin, the structures and binding site sites of the actin crosslinking proteins that form the actin bundles that support the microvilli of the brush border epithelium have been identified and modeled. The headpiece domain is a modular, 76-amino acid F-actin binding motif that provides the essential second actin-binding site that allows villin to crosslink actin filaments into structural bundles. Headpiece domains are also found on at the C-termini of several classes of large "core" domains unrelated to villin. The focus of this proposal is on determining how and where headpiece domains bind actin filaments, to produce a model of the headpiece-actin complex, and to investigate the flexibility of sequence linking villin headpiece to its core domain. The broad, long-term objectives of this proposal are to determine at the molecular level how actin bundles like those that support the microvilli of brush border epithelial cells are assembled and organized. The specific aims of this proposal are: 1) To determine the high resolution structure of villin headpiece and the NMR structures the phosphoryl-regulated dematin headpiece, and the non-actin binding supervillin headpiece. 2) To test the published "hydrophobic cap, charged crown, and basic patch" hypothesis of headpiece-F-actin recognition by mutagenesis of the villin and supervillin headpiece domains 2. To locate the binding site of the headpiece motif on F-actin and computer models of the F-actin headpiece complex. 3. To determine the flexibility of the linkage between villin headpiece and its gelsolin- like core by 15N-NMR relaxation measurements. The health relatedness of this project is that by understanding how essential actin bundles, like those that support the absorptive epithelial brush border, are organized in the cell we may be able to manipulate their formation in epithelial and other cell types.